The present invention provides a method for the identification of a pattern of changes in cellular responses induced by the selective inhibition of a signaling molecule, and methods for identifying selective inhibitors thereof.
Cell-to-cell communications in a multicellular organism are fast and allow cells to respond to one another in diverse and complex ways. Typically, the intracellular signals are molecules called xe2x80x9cligands,xe2x80x9d and a given ligand can bind to a particular type of receptor on the surface of those cells that are to receive that signal, but this simple ligand binding alone is not enough to provide for the complex responses that the receiving cells may need to make. Cells therefore amplify and add complexity to this signal through complex, often cascading, mechanisms leading to the rapid modulation of catalytic activities inside the cell, which in turn can produce complex, and sometimes dramatic, intracellular responses. This process as a whole, from initial ligand binding to completion of the intracellular response, is called xe2x80x9csignal transduction.xe2x80x9d
Signal transduction is often accomplished by the activation of intracellular enzymes that can act upon other enzymes and change their catalytic activity. This may lead to increases or decreases in the activity certain metabolic pathways, or may lead to even large intracellular changes, for example, the initiation of specific patterns of gene expression. The ability of one enzyme to alter the activity of other enzymes generally indicates that the enzyme is involved in cellular signal transduction.
The most common covalent modification used in signal transduction processes is phosphorylation, which results in the alteration of the activity of those enzymes which become phosphorylated. This phosphorylation is catalyzed by enzymes known as ATP-dependent phosphotransferases which are often simply referred to as xe2x80x9ckinases.xe2x80x9dThese include, among others, protein kinases, lipid kinases, inositol kinases, non-classical protein kinases, histidine kinases, aspartyl kinases, nucleoside kinases, and polynucleotide kinases.
Several key features of such kinases make them ideally suited as signaling proteins. One is that they often have overlapping target substrate specificities, which allows xe2x80x9ccross-talkxe2x80x9d among different signaling pathways, thus allowing for the integration of different signals (1). This is thought to be a result of the need for each kinase to phosphorylate several substrates before a response is elicited, which in turn provides for many types of diverse signaling outcomes. For example, a given kinase may in one instance transmit a growth inhibitory signal and in another instance transmit a growth promoting signal, depending on the structure of the extracellular ligand that has bound to the cell surface (2).
A second key feature is that the kinases are organized into several modular functional regions, or xe2x80x9cdomainsxe2x80x9d (3). One domain known as xe2x80x9cSH3xe2x80x9d is a proline-rich region of 55-70 amino acids in length, and another, known as xe2x80x9cSH2,xe2x80x9d is a phosphotyrosine-binding region of about 100 amino acids in length. These two domains are believed to be involved in recognizing and binding to the protein substrates. The third domain, xe2x80x9cSH1,xe2x80x9d is comprised of about 270 amino acids, and is the domain which is responsible for catalysis. It also contains the binding site for the nucleoside triphosphate which is used as energy source and phosphate donor (3). Other domains, including myristylation and palmitylation sites, along with SH2 and SH3, are responsible for assembling multiprotein complexes which guide the catalytic domain to the correct targets (3,22,23). Molecular recognition by the various domains has been studied using by x-ray diffraction and by using NMR methods (24-28).
These domains appear to have been mixed and matched through evolution to produce the large protein kinase xe2x80x9cfamily.xe2x80x9d As many as 2000 kinases are thought to be encoded in the mammalian genome (4), and over 250 kinases have already been identified. The large number of kinases and the large number of phosphorylation-modulated enzymes that are known to exist inside cells allow for rapid signal amplification and multiple points of regulation.
A third key feature of the kinases is their speed. The kinetics of phosphorylation and dephosphorylation is extremely rapid in many cells (on a millisecond time scale), providing for rapid responses and short recovery times, which in turn makes repeated signal transmission possible (5).
These features of the kinases have apparently led them to be involved in a vast array of different intracellular signal transduction mechanisms. For example, growth factors, transcription factors, hormones, cell cycle regulatory proteins, and many other classes of cellular regulators utilize tyrosine kinases in their signaling cascades (12,13). Tyrosine kinases catalytically attach a phosphate to one or more tyrosine residues on their protein substrates. The tyrosine kinases include proteins with many diverse functions including the cell cycle control element c-abl (14-16), epidermal growth factor receptor which contains a cytoplasmic tyrosine kinase domain (12),c-src, a non-receptor tyrosine kinase involved in many immune cell functions (13), and Tyk2, a cytoplasmic tyrosine kinase which is involved in phosphorylation of the p91 protein which is translocated to the nucleus upon receptor stimulation and functions as a transcription factor (17).
The serine/threonine kinases make up much if not all of the remainder of the kinase family; these catalytically phosphorylate serine and threonine residues in their protein substrates, and they have similarly diverse roles. They share homology in the 270 amino acid catalytic domain with tyrosine kinases. As such, although the discussion which follows focuses more particularly on the tyrosine kinases, that discussion is generally applicable to the serine/threonine kinases as well. An example of a protein kinase in yeast is CDC28. This is the major protein kinase in yeast which controls the cell cycle.
One important avenue for deciphering the role and understanding the function of enzymes, both in vitro and in vivo, is the use of specific enzyme inhibitors. If one or more compound can be found that will inhibit the enzyme, the inhibitor can be used to modulate the enzyme""s activity, and the effects of that decrease can be observed. Such approaches have been instrumental in deciphering many of the pathways of intermediary metabolism, and have also been important in learning about enzyme kinetics and determining catalytic mechanisms.
In addition, such inhibitors are among the most important pharmaceutical compounds known. For example, aspirin (acetylsalicylic acid) is such an inhibitor. It inhibits an enzyme that catalyzes the first step in prostaglandin synthesis, thus inhibiting the formation of prostaglandins, which are involved in producing pain (72). Traditional drug discovery can be characterized as the design and modification of compounds designed specifically to bind to and inactivate a disease-causing protein; the relative success of such an effort depends upon the selectivity of the drug for the target protein and its lack of inhibition of non-disease associated enzymes with similar enzyme activities.
Such approaches would appear to be promising ways to develop treatments for cancer, since many human cancers are caused by dysregulation of a normal protein (e.g., when a proto-oncogene is converted to an oncogene through a gene translocation). And since kinases are key regulators, they have turned out to be very common proto-oncogenes, and thus ideal drug design targets. The process of designing selective inhibitors is relatively simple in cases where few similar enzymes are present in the target organism, for example in cases where inhibitors of a protein unique to bacteria can be targeted. But unfortunately, the similarities between the kinases and their large number has frustrated the discovery and design of specific inhibitors, and has blocked most hopes of developing specific pharmaceutical treatments aimed at the proto-oncogene level. It is expected that the vast majority of candidate inhibitors will inhibit multiple kinases, even though they may have initially been identified as inhibiting a particular, purified kinase.
This is not to say, however, that inhibitors with at least some degree of kinase-specificity cannot be found. Several natural products have been identified which are relatively specific for particular kinase families, but attempts to derive general rules about kinase inhibition based on these has failed. Furthermore, as the following examples show, specificity in most cases is quite limited. For example, the compound damnacanthal was reported to be a xe2x80x9chighly potent, selective inhibitorxe2x80x9d of the kinase p56lck (73); this compound has an 50% inhibitory concentration (IC50) for that kinase which is almost seven times lower than for the kinase src (the IC50 is the concentration of inhibitor which must be added to reduce catalytic activity by 50%). The compound PPI has a binding affinity for the kinase lck which is very strong (IC50=0.005 xcexcM); but unfortunately, the inhibition of other kinases of the src family is very similar. It inhibits the kinase fyn with an almost identical IC50, 0.006 xcexcM, and has only about a 4-fold higher IC50 for the kinase hck (IC50=0.020 xcexcM). The compound CGP 57148 has been reported to be xe2x80x9csemi-selectivexe2x80x9d for the kinases abl (IC50=0.025 xcexcM) and PDGFR (IC50=0.030 xcexcM)(74). Nevertheless, considering the vast number of kinases and their relative cellular importance, and also considering that the above-described inhibitors have only been reported in the last two years, it appears that success in discovering or designing selective kinase inhibitors has been limited.
These difficulties described above have implications well beyond the mere frustration of scientists; they have frustrated efforts to decipher the kinase cascades and the function of individual kinases in those cascades and other cellular mechanisms. Such an understanding of kinase activity and function may be essential before certain human diseases can be effectively treated, prevented or cured. For example, it has been known for over 30 years that the oncogene bcr-abl is a protein kinase that is responsible for chronic myelogenous leukemia; but the physiological substrates that it acts upon to cause oncogenesis, which may be important drug design targets, have yet to be definitively identified (11). On the bright side, despite this shortcoming, the above-described inhibitor CGP 57148 is reportedly now undergoing clinical trials for use in treating myelogenous leukemia, even though the substrates it may block phosphorylation of in vivo are not known.
The medical significance of these difficulties is further illustrated by the Rous sarcoma virus (RSV), which has become an important model system for studying the role of kinases in oncogenesis. RSV transformation of fibroblasts is controlled by a single viral gene product, the protein tyrosine kinase v-src (32). It is the rapid time course and the dramatic morphological changes during RSV fibroblast transformation that have made RSV a paradigm for studies of oncogene activity in all cells. The origin (33), regulation (3,8,34,35), and structure (25,27,36) of v-Src have been extensively studied and are well understood (8,37,38). But central questions about this intensely studied kinase remains unanswered: what are its direct cellular substrates? Does inhibition of its catalytic activity effectively inhibit, or even reverse, transformation? Would such inhibition be an effective therapy for or prophylactic against RSV transformation? Unfortunately, as discussed above, the answers to these questions are not forthcoming, largely because the number of cellular kinases is enormous (it is estimated that 2% of the mammalian genome encodes protein kinases (4)) and because tyrosine kinases display overlapping substrate specificities (8,39) and share catalytic domains, making the design of specific inhibitors difficult.
The expression of v-Src in fibroblasts results in the tyrosine phosphorylation of over 50 cellular proteins (37). These same substrates are also phosphorylated by other kinases in untransformed fibroblasts (40). Even sophisticated biochemical and genetic techniques, including anti-phosphotyrosine protein blots of transformed fibroblasts, transfection of fibroblasts with transformation-defective v-Src mutants, temperature-sensitive v-Src mutants, gene knock-out studies of cellular Src, host-range dependent Src mutants, anti-v-Src immunoprecipitation, and use of kinase-specific inhibitors, have not led to the unambiguous identification of v-Src""s direct substrates (see reference (38) for a comprehensive review). But this situation is not unique; in fact, the direct substrates for the majority of cellular kinases remain unidentified (8). Furthermore, as discussed above, there also are few compounds known to selectively inhibit individual kinases, or even groups of related kinases.
Certain protein kinases because of their central importance in cell division have become very active drug targets. One such kinase is cyclin dependent kinase 2 (the yeast homolog is CDC28). CDK2 activity is required to proceed through the cell cycle. Therefore, diseases which are caused by abnormal growth of cells or tissues could be blocked if the progression through the cell cycle could be blocked by inhibition of CDK2 in these cells. Examples of such diseases include: tumor growth, restenosis and atherosclerosis, glomerulonephritis, psoriasis, and Alzheimer""s disease. The basis for disease treatment is that disease related cells must divide faster than xe2x80x9cnormalxe2x80x9d cells and thus will be more sensitive to potent agents which block cell cycle progression.
In particular, cell cycle control kinases are important targets for anti-cancer therapy based on the identification of the role of these kinases in controlling cellular proliferation. One such example is the yeast homolog CDC28 of such a cell cycle control kinase.
From the forgoing, it is clear that there has been a long felt but unsatisfied need for ways to identify inhibitors of specific signaling molecules, protein kinases being an example. More particularly, a need exists for a method for identifying specific inhibitors without the arduous task of expression, purification, and assay of the ever-growing number of described signaling molecules, a number that is increasing rapidly. Over 1000 kinases are known. The need exists for method for identifying specific inhibitors of individual kinases or kinase families, which could be used to identify protein substrates (by looking for which proteins are not phosphorylated or are more weakly phosphorylated in the presence of the inhibitor), to study the biochemical and phenotypic effects of rapidly down-regulating a given kinase""s activity, for use as drugs to treat kinase-mediated diseases, and to confirm that tedious efforts to design or develop more traditional inhibitor drugs would be worthwhile.
It is towards the development of a method for identifying a pattern of cellular responses resulting from the selective inhibition of a signaling molecule, and methods of using the pattern for identifying selective inhibitors of the signaling molecule, that the present invention is directed.
The citation of any reference herein should not be construed as an admission that such reference is available as xe2x80x9cPrior Artxe2x80x9d to the instant application.
The following terms have the definitions as used herein:
xe2x80x9cSignaling Moleculexe2x80x9d is a molecule involved in transmitting information about the external environment surrounding a cell: e.g., a receptor for a growth factor, or an enzyme which is activated after the growth factor binds to the receptor.
xe2x80x9cCellular Responsexe2x80x9d is any quantitatively or qualitatively measurable change of a unitary or complex event in a cell, for example, as a consequence of exposure to a stimulant, an inhibitor, change in the environment, or change over time. Such a cellular response may be either decreased, increased, or unchanged, compared to that which would occur in the absence of such exposure, such as increased, decreased or unchanged levels of gene transcription, protein expression, metabolism, etc. over a particular time period, or as a consequence of a perturbation to the cell, whether environmental, chemical, or otherwise. The particular cellular response(s) need not be further characterized, for example, as to specific gene transcripts or proteins.
xe2x80x9cPattern of Cellular Responsesxe2x80x9d is a composite representation of the changes or lack of changes in a plurality of cellular responses characteristic of or attributable to the inhibition of a particular signaling molecule in a cell.
xe2x80x9cAlteredxe2x80x9d refers to a change in the level of a cellular response. This change may be but is not limited to increased, decreased, disappearance, or appearance.
xe2x80x9cWild typexe2x80x9d refers to the naturally occurring (non-mutated) form of the signaling molecule under consideration. The wild-type form of the signaling molecule may be provided in a host cell, referred to herein as the wild-type cell. This cell may or may not be the natural cell in which the signaling molecule is present.
xe2x80x9cMutantxe2x80x9d refers to an form of the signaling molecule containing one or more changes to the protein sequence of the wild-type sequence. A functionally silent mutation refers to a mutation which does not alter the phenotype of the cell, i.e., the signaling molecule for which the mutant gene encodes retains its function as compared to the wild-type molecule. The mutant form of the molecule is provided in a host cell, referred to herein as the mutant cell.
The present invention provides methods by which the pattern of cellular responses by the selective inhibition of a signaling molecule may be determined, and using this pattern, selective inhibitors of the signaling molecule may be identified. It has been discovered by the inventors herein that a characteristic pattern of cellular responses that is determined from the selective inhibition of a functional, silent mutation of a particular signaling molecule, is a pattern that represents the cellular responses resulting from the inhibition of the wild-type signaling molecule. Thus, a pattern of cellular responses identified for a particular signaling molecule may be used to identify selective inhibitors by determining whether the characteristic pattern results from the candidate inhibitor; furthermore, the pattern of effects of an inhibitor may be used to identify the signaling molecule being inhibited.
In its broadest aspect, the present invention is directed to patterns of cellular responses attributable to the selective inhibition of a wild-type form of a preselected signaling molecule, wherein the patterns comprise changes in the cellular responses attributable to selective inhibition of a mutant form of the preselected signaling molecule by a selective inhibitor of the mutant form of the preselected signaling molecule. The invention is directed further to the use of the aforementioned patterns to identify selective inhibitors of wild-type forms of signaling molecules.
In another aspect, the present invention involves methods for identifying a pattern of cellular responses attributable to the selective inhibition of a wild-type form of a preselected signaling molecule comprising the steps of:
A method for identifying a pattern of cellular responses attributable to the selective inhibition of a wild-type form of a preselected signaling molecule comprising the steps of:
(a) providing mutant cells which have a mutant signaling molecule in place of or coexisting with the wild-type signaling molecule;
(b) providing a selective inhibitor of the mutant signaling molecule;
(c) identifying cellular responses exhibited by the mutant cells before and after exposure to the mutant inhibitor, optionally in addition to identifying the exhibited cellular responses selected from
i) wild-type cells unexposed to said mutant inhibitor,
ii) wild-type cells after exposure to said mutant inhibitor, or
iii) the combination of i) and ii) above; and
(d) comparing said cellular responses obtained in step (c) above to identify a pattern of cellular responses attributable to the selective inhibition of said wild-type preselected signaling molecule wherein the pattern comprises the cellular responses attributable to selective inhibition of the mutant signaling molecule in the mutant cells.
In a further aspect, the present invention involves methods for identifying a pattern of cellular responses attributable to the selective inhibition of a wild-type form of a preselected signaling molecule comprising the steps of:
(a) providing wild-type cells which have a wild-type signaling molecule,
(b) providing mutant cells which have a mutant signaling molecule in place of or coexisting with the wild-type signaling molecule;
(c) providing a selective inhibitor of the mutant signaling molecule;
(d) identifying cellular responses exhibited by wild-type cells that are not exposed to the mutant inhibitor;
(e) identifying cellular responses exhibited by wild-type cells after exposure to the mutant inhibitor;
(f) identifying cellular responses exhibited by the mutant cells which are not treated with the mutant inhibitor;
(g) identifying cellular responses exhibited by the mutant cells after exposure to the mutant inhibitor; and
(h) comparing said cellular responses in steps (d), (e), (f) and (g) to identify a pattern of cellular responses attributable to the selective inhibition of said wild-type preselected signaling molecule wherein said pattern comprises the cellular responses attributable to selective inhibition of said mutant signaling molecule in said mutant cells.
In a further embodiment, the wild-type and mutant cells in either of the foregoing methods may be exposed to a stimulant in order to induce cellular responses. Non-limiting examples of such stimulants include hormones, cytokines, growth factors, heat, cold, light, metal ions, osmolarity changes, contact, heterologous cells, pressure, oxidative stress, natural products, plant extracts, marine organisms, synthetic compounds, combinatorial organic libraries, peptide libraries, organ tissue explants, or via cell transfer into animals, among other factors, which will be related to the particular cell type.
The foregoing method is applicable to a wide variety of signaling molecules. By way of non-limiting examples, such molecules as transcription factors, ATP-dependent phosphotransferases, myosin motors, histone acetyl transferases, ion channels, farnesyl transferases, ligand gated channels, metabolic enzymes, natural product targets, the proteosome, ubiquitin pathway enzymes, complement system enzymes, proteases, intracellular stores of ions, vesicle trafficking enzymes, G-protein coupled receptors, proteases, and other signal transduction molecules are applicable to the present invention. In a preferred embodiment, the signaling molecule is an ATP-dependent phosphotransferase, such as a protein kinase, lipid kinase, inositol kinase, non-classical protein kinase, histidine kinase, aspartyl kinase, nucleoside kinase, or polynucleotide kinase. Non-limiting examples of protein kinases include those in groups known AGCs, calmodulin dependent protein kinases, CMGCs, protein tyrosine kinases, or other protein kinases. In a preferred embodiment, the protein kinase is CDC28 in yeast or v-src and cdk2 in humans.
The cellular responses identified in the foregoing method include any quantitatively or qualitatively measurable appearance, change, or disappearance of a parameter such as but not limited to gene transcription, protein expression, metabolic alteration, morphologic alteration, lipid alteration, growth alteration, cell shape change, cytoskeletal reorganization, protein translocation, protein relocalization, metal ion influx, metal ion efflux, change in osmolarity, receptor expression on the cell surface, receptor clustering, receptor desensitization, protein glycosylation, protein destruction, protein phosphorylation or other protein post-translational modification. The pattern of cellular changes may be a single or a plurality of changes, for example, a morphological change or the expression of a particular protein or mRNA. The pattern may be a plurality of responses, such as changes in a number of individual gene transcription products, perhaps hundreds to thousands; a plurality of proteins expressed, the levels of various intracellular metabolites of secreted products; the lipid makeup of the cell membrane, and so forth. These responses may be measured by known techniques applicable to the particular type of change. For example, changes in gene transcription may be measured using DNA chip array technology, cDNA array techniques on glass or nitrocellulose filters, oligonucleotide arrays on various solid supports, TAQman assay, quantitative PCR, competitive PCR, and differential display. Certain of these techniques may be better applicable to measuring a large number of responses, such as the DNA chip array technology for a large number of individual mRNA transcripts. By way of another example, a large number of expressed proteins may be measured using differential display, 2-D protein gel electrophoresis, mass spectroscopy, high-throughput mass spectroscopy, massively parallel protein identification technologies such as those based on monoclonal or polyclonal antibody recognition, RNA or DNA polymers which recognize various proteins, such RNA or DNA molecules could be produced from so-called in vitro evolution experiments, high-throughput confocal microscopy, X-ray diffraction, nuclear magnetic resonance spectroscopy, resonance Raman spectroscopy, capillary electrophoresis, and so forth.
The wild-type cells may be derived from any organism. This includes eukaryotic or prokaryotic cells. Eukaryotic cells include for example plant cells, animal cells such as mammalian, including human cells, and protistan cells including yeast, protozoans, and other eukaryotic microorganisms. Prokaryotic cells include bacteria, blue-green bacteria, including members of the Archaebacteria and eubacteria. The wild-type cells may be hosts for a signaling molecule from another species or kingdom.
The mutant cells with the mutant form of the signaling molecule may be prepared by any one of a number of methods known to the skilled artisan. For example, the mutant cells may be prepared by gene knock-in technology; or by mating or fusion between an organism or cell lacking the wild-type kinase and an organism or cell which contains both the wild type and the mutant kinase gene, followed by screening the progeny or resultant fusions for those which only have the mutant kinase gene and lack the wild-type kinase gene mutation. Furthermore, by way of non-limiting example, the methods described in copending application Ser. No. PCT/US98/02522 (published as WO 98/35048), incorporated herein by reference, may be used. The mutant signaling molecule may be of origin from any species or kingdom, and provided in any suitable host cell or any species or kingdom, to provide a mutant cell with the mutant signaling molecule suitable for use for the methods herein.
Selective inhibitors of the mutant signaling molecule may be identified and prepared by any of a number of methods known to the skilled artisan. Furthermore, by way of non-limiting example, the methods described in copending application Ser. No. PCT/US98/02522 (published as WO 98/35048), and in copending application Serial No. 60/115,340, incorporated herein by reference, may be used. By way of non-limiting example, the inhibitor 4-amino-1-(tert-butyl)-3-(1xe2x80x2-naphthylmethyl)(tert-butyl)-3-(1xe2x80x2-naphthylmethyl)pyrazolo[3,4-d]pyrimidine is useful for the practice of the present invention.
For example, the pairs of wild type and mutant genes that may be used in the method of the present invention include but are not limited to CDC28 and CDC28 F88G, v-Src and v-Src 1338G, c-AMP dependent kinase (PKA) and PKA M120G or PKA M120A, p38 and p38 T106A or p38 T106G, Raf and Raf (V420A) or Raf (V420G), and the insulin receptor kinase (IRK) IRK(V1075A) or IRK (V1075G).
By way of example, a signaling molecule that may be selected to identify the characteristic pattern of cellular response to its inhibition in accordance with the present invention may be the yeast protein kinase CDC28, the mutant cells known as CDC28 expressing a mutant form of CDC28 referred to as CDC28 F88G, the selective inhibitor of the mutant inhibitor is 4-amino-1-(tert-butyl)-3-(1xe2x80x2-naphthylmethyl)pyrazolo[3,4-d]pyrimidine, and the cellular responses are gene transcription products. The gene transcription products are measured using DNA chip array technology.
In a further aspect of the present invention, the aforementioned method comprising the exposing the wild-type cells and the mutant cells to a non-specific inhibitor, and the pattern of cellular responses additionally comprising cellular responses which are altered or unaltered by the non-specific inhibitor. Non-limiting examples of non-specific inhibitors include 4-amino-1-tert-butyl-3-(p-methylphenyl)pyrazolo[3,4-d]pyrimidine, genestein, quercetin, K252a, staurosporine, adenosine, olomoucine, SKB 203580, damnacanthal, tyrphostins, erbstatin, piceatannol, lavendustin A, and radicicol.
In another broad aspect of the present invention, methods are provided for identifying a selective inhibitor of a wild-type form of a preselected signaling molecule. The method comprises the steps of first identifying a pattern of cellular responses attributable to the selective inhibition of a wild-type form of the preselected signaling molecule in accordance with the above-described methods; exposing wild-type cells to a candidate selective inhibitor of the wild-type form of the signaling molecule; identifying the effect of the candidate inhibitor on the pattern of cellular responses in the wild-type cell; and identifying a selective inhibitor as that which matches or resembles the pattern. In a further aspect, the wild-type cells are additionally exposed to a stimulant to induce the cellular responses, as described above.
These and other aspects of the present invention will be better appreciated by reference to the following drawings and Detailed Description.